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Protein Splicing: Mechanism and Applications in Bioorganic Chemistry

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Protein Splicing: Mechanism and Applications in Bioorganic Chemistry PROTEIN SPLICING: MECHANISM AND APPLICATIONS IN BIOORGANIC CHEMISTRY Reported by Kimberly L. Berkowski February 19, 2001 INTRODUCTION The discovery of protein splicing has altered the central dogma of gene expression.1 According to this dogma, genetic information flows from DNA to RNA through transcription, and is then translated and expressed as protein. However, more genetic information is typically coded for than actually appears in the protein product. This information is DNA posttranscriptionally excised as introns, in a process Transcription termed RNA splicing. Just over ten years ago, two groups independently discovered that some proteins RNA are capable of excising genetic information Translation posttranslationally through a process analogous to N-extein 2,3 RNA splicing. In protein splicing internal intein segments in the posttranslational product (inteins) Protein Precursor excise themselves from the protein while ligating C-extein the flanking polypeptides (exteins) to form the final Protein splicing protein product (Figure 1). This excision and Functional Protein ligation process is novel because it is self- + 4 catalyzed. The discovery of protein splicing and elucidation of its mechanism triggered the research Ligated Extein Excised Intein of new applications and techniques for protein Figure 1. Protein Biosynthesis. chemistry and engineering. INTEINS: OCCURRENCE AND STRUCTURE To date, over 100 inteins have been discovered - all from proteins of unicellular organisms.5 Approximately 70% of inteins reside in host proteins involved in DNA replication and repair. The reasons for this intein preference are unclear, although many hypotheses have been proposed.6 Inteins have been found to contain 134-608 amino acids. They are usually composed of two distinct regions: a protein splicing domain (which is split into two segments) and an endonuclease domain.5 Smaller inteins, however, contain only the split protein splicing domain separated by a short amino acid linker Copyright © 2001 by Kimberly L. Berkowski 1 (Figure 2).7 The protein splicing domain catalyzes its own excision from the protein as well as the ligation of the flanking exteins. The endonuclease region is a mobile genetic element that duplicates itself and inserts into a homologous, but intein-lacking allele.6 The two domains of the intein function independently of each other.7 This review will focus only on the protein splicing region of the intein. A B G Splicing domain Endonuclease domain Splicing domain N-extein C-extein A B G Splicing domain Linker Splicing domain N-extein C-extein Figure 2. Intein Structure. Certain amino acids are highly conserved within the protein splicing domain. The first amino acid on the N-terminal intein (block A) and the C-terminal extein is either serine, cysteine, or threonine, although an alanine residue has sometimes been observed at the N-terminal splice junction.1 The amino acid on the C-terminal of the intein (block G) is most commonly asparagine, preceded by a histidine.7 Finally, a Thr-x-x-His sequence in block B of the N-terminal intein is usually observed (Figure 2).1 These residues play specific roles in the structure of the intein and the mechanism of protein splicing as discussed below. MECHANISM Protein splicing occurs via four independent,4 intramolecular8 reactions. The first three reactions are catalyzed by the intein at a single site.7 The final reaction is a spontaneous rearrangement to form a native peptide bond. Although the chemical reactions involved in protein splicing are well understood, the mechanism of catalysis for each reaction is just now beginning to be elucidated. N ! X Acyl Rearrangement The first step of protein splicing involves the nucleophilic attack of the first intein residue, the conserved cys/ser amino acid, on the adjacent carbonyl carbon of the extein (Figure 3). The reaction goes through an oxyoxazolidine or oxythiazolidine anion to form 1, an ester or thioester.1 Because the equilibrium constant is typically unfavorable for the rearrangement of an amide to an ester or thioester, the peptide bond at the N-terminal splice junction has been proposed to exist in a high energy conformation.7 The crystal structures of a mutant GyrA protein from Mycobacterium xenopi and a mutant VMA protein from Saccharomyces cerevisiae (VMA29) support this hypothesis. The crystallized GyrA intein has one N-terminal extein residue, 197 XH O N-extein H intein intein residues, and no C-terminal N N C-extein H O H2N extein residues. In addition, the first XH O Step 1: N-X acyl rearrangement intein residue was mutated from cysteine to serine to retard splicing.9 In the crystallized VMA29 protein, the XH NH2 O N-extein cysteine residue at the N-terminal X intein N C-extein O H splice junction, and the asparagine H2N Step 2: Transesterification residue at the C-terminal splice O 1 junction were mutated to alanine to prevent splicing. The N-terminal extein contains 10 amino acid residues N-extein O HX X and the C-termial extein contains four O H2N intein N amino acid residues. The crystal H C-extein Step 3: Asparagine Cyclization NH2 structure of the GyrA intein shows the O 2 scissile peptide bond at the N-terminal splice junction in a less energetically favorable cis conformation.9 This O HX C-extein O N-extein X bond conformation correctly positions H2N intein NH2 NH the serine residue at the N-terminal 3 Step 4: X-N acyl rearrangement O splice junction for nucleophilic attack 4 XH on the adjacent carbonyl carbon of the O 9 extein. The crystal structure of N-extein N C-extein H VMA29 however, indicates that a Figure 3. Mechanism. 5 distorted trans conformation exists at the N-terminal splice junction.10 These crystal structures support the hypothesis that ester formation may occur to relieve the strain of a high energy peptide bond. The crystal structures of the GyrA and VMA29 inteins also help to explain the unfavorable amide-ester equilibrium shift by showing amino acid residues that could potentially stabilize the tetrahedral intermediate through hydrogen bonding via an oxyanion hole.10 Stabilization of the tetrahedral intermediate lowers the activation energy of the reaction and facilitates ester formation. Finally, both crystal structures show a conserved histidine residue positioned within hydrogen bonding distance to the nitrogen of the scissile peptide bond. This histidine residue may catalyze the reaction by donating a proton to the amine, making it a better leaving group. Although much insight has been recently obtained for the catalysis of the N ! X acyl rearrangement, the mechanism remains somewhat ambiguous. Transesterification In the second step of protein splicing, the conserved cys/ser/thr amino acid on the C-terminal extein of 1 attacks the newly formed ester group of the N-terminal splice junction. This transformation results in the ligation of the exteins as shown by 2. The crystal structure of VMA29 shows that a 9 Å gap exists between the nucleophile and electrophile.10 How this gap is transversed by the nucleophilic residues is unclear. However, Poland and co-workers propose that a conformational change after the first step brings the splice junctions into closer proximity. The crystal structure of the VMA29 intein shows the presence of a zinc ion at the C-terminal splice junction. The zinc ion is coordinated to the nucleophilic cysteine (Cys455) involved in transesterification, the penultimate histidine residue (His453), a glutamic acid (Glu80), and a water molecule. This coordination strains the backbone of the protein and 10 may facilitate transesterification. In addition, Shingledecker and coworkers determined that the pKa of 455 the sulfhydryl group on Cys is 5.8. Typically, the sulfhydryl groups in proteins have pKa values of 8. Thus, this result correlates well with the discovery of the coordination of cysteine to zinc. Asparagine Cyclization In the third step of protein splicing the amide side chain of asparagine at the C-terminal splice junction attacks its own carbonyl carbon on the main chain. The asparagine residue cyclizes, forming a C-teminal aminosuccinimide, 3, and the excised intein 4.8 The crystal structure of the VMA29 intein has provided insight to the mechanism and catalysis of asparagine cyclization. Poland and coworkers suggest that transesterification to form the branched intermediate disrupts the coordination between the cysteine residue and the zinc ion. As a result zinc may move, coordinate to His442 and one of amino acid residues flanking Cys455. As a result, the penultimate histidine (His453) may move to a position where it is able to protonate the main chain amide of Asn454 after cyclization, catalyzing peptide hydrolysis. In addition, the reorientation of zinc as well as some hydrophobic interactions may correctly position the β-amide of asparagine to attack its backbone carbonyl carbon.10 X ! N Acyl Rearrangement In the final step of protein splicing the ester moiety rearranges to form a protein with a more thermodynamically stable amide group, 5. Unlike the first step of protein splicing, an intein is not present to stabilize the ester group. Therefore, this step is spontaneous and virtually irreversible.1 APPLICATIONS The discovery that inteins self-catalyze their excision from proteins has led to the development of interesting and useful applications in protein science and engineering. Two general types of applications stem from protein splicing. The first application involves the mutation of either the N- or C-terminal splice junction to inhibit protein splicing. Controlled cleavage is still allowed, however, at the unmutated splice junction. In the second application, protein splicing is induced by the reassembly of a split intein. Intein-Mediated Affinity Chromatography for Protein Purification Intein-mediated protein purification is a common application of protein splicing. With this technique, specific reagents, temperature, or pH are used to controllably cleave the intein at either its N- or C-terminal splice junction.13,14 Intein-mediated protein purification systems work by fusing one terminus of the intein to an affinity tag and the other terminus to a target protein.
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